Application of eucommia ulmoides extract in preparation of medicine for treating cisplatin renal injury

Abstract

The invention discloses application of eucommia ulmoides extract in preparation of a medicine for treating cisplatin renal injury, and relates to the technical field of biological medicine. The preparation method comprises the following steps: firstly, preparing the eucommia ulmoides extract, performing ethanol reflux extraction on eucommia ulmoides bark to obtain an extracting solution, and adsorbing and purifying the extracting solution to obtain the eucommia ulmoides extract. The eucommia ulmoides extract can significantly improve the activity of cis-platinum induced HK2 cells; the HK2 cell shrinkage damage is relieved; the relative expression level of a cisplatin-induced renal injury marker KIM-1 is also down-regulated; the eucommia ulmoides extract can also significantly reduce plasma creatinine, blood urea nitrogen and ALT levels of an acute kidney injury mouse model, and improve cisplatin-induced acute kidney injury; meanwhile, the eucommia ulmoides extract and cis-platinum are combined for use, so that the kidney injury can be improved, the effect of inhibiting endometrial cancer cell Ishikawa cells is also achieved, and a very good synergistic anti-tumor cell effect is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and particularly relates to application of an eucommia ulmoides extract in preparing a medicine for treating cisplatin-induced kidney injury. Background Art

[0002] Cisplatin (CP) is a highly effective first-line anti-tumor drug, widely used in the treatment of various solid malignancies such as lymphoma and endometrial cancer. According to statistics, about 50% of cancer patients use cisplatin in their chemotherapy regimens. The clinical use of cisplatin is accompanied by the occurrence of a variety of adverse reactions, mainly including nephrotoxicity, ototoxicity, gastrointestinal toxicity, neurotoxicity, etc. Among them, nephrotoxicity is considered to be a potentially irreversible serious adverse reaction, which seriously limits the clinical use of cisplatin. The accumulation of cisplatin in the kidneys can lead to acute and chronic nephrotoxicity. During the treatment of cisplatin, about 40% of patients cannot complete the treatment due to renal damage. The mechanism and prevention and treatment measures of cisplatin renal injury have always been an important topic of concern in the medical and pharmaceutical communities. The molecular mechanism of cisplatin-induced renal injury is that the uptake of cisplatin in renal proximal tubular cells is mediated by membrane transporters, such as solute carrier family 22 member 2 (SLC22A2). After cisplatin enters the cell, chloride ions are replaced by water molecules. Hydrated cisplatin is metabolically activated to a toxic form through a γ-glutamyltranspeptidase (GGT) and kynurenine aminotransferase 1 (KYAT1)-dependent pathway. Accumulation of cisplatin in renal proximal tubular cells can induce processes such as DNA damage, mitochondrial damage, oxidative stress, endoplasmic reticulum stress, autophagy, and cell cycle regulation. These processes lead to renal cell death, inflammation, and cell senescence, leading to acute kidney injury (AKI) and chronic kidney disease (CKD). Therefore, it is crucial to find or develop drugs that can treat cisplatin-induced renal injury.

[0003] Eucommia ulmoides Oliver (Euo), also known as Si Zhong, is one of the traditional Chinese herbal medicines in my country. It has a history of more than 2,000 years of use in my country. It is a unique precious medicinal material and economic tree species in my country, and is also an important strategic reserve resource in my country. Many ancient medical books record that Eucommia ulmoides has the effect of tonifying the kidney, such as "Shennong's Herbal Classic": it treats low back pain, replenishes the middle and benefits the essence; "Compendium of Materia Medica": it treats low back pain, replenishes the middle and benefits the essence; "Shui Yao Yao": the stem bark is used to lower blood pressure and nephritic edema; "Gui Yao Bian": the bark treats kidney deficiency and low back pain. Due to its complex chemical composition, the pharmacological effects of Eucommia ulmoides have attracted much attention. Eucommia ulmoides extract has a variety of natural bioactive ingredients, mainly lignans, cyclopentane ether terpenes, flavonoids, phenolic acids, steroid compounds, antifungal proteins, etc. Modern pharmacological studies have found that it has pharmacological properties such as neuroprotection, anti-tumor, anti-inflammatory, antioxidant, blood pressure reduction, blood lipid regulation, and blood sugar reduction. Our team's early research confirmed that the active ingredients in Eucommia extract can improve diabetic kidney damage. Its mechanism of action is that Eucommia extract improves cell damage and mitochondrial dysfunction through the AR / Nrf2 / HO-1 / AMPK axis, thereby effectively improving diabetic nephropathy (Huang, Qi et al. Eucommia lignans alleviate the progression of diabetic nephropathy through mediating the AR / Nrf2 / HO-1 / AMPKaxis in vivo and in vitro. Chinese journal of natural medicines vol. 21,7(2023): 516-526). So far, there have been no reports on the protective effect of Eucommia extract on cisplatin-induced acute kidney injury. Summary of the invention

[0004] The purpose of the present invention is to provide an application of eucommia extract in the preparation of a drug for treating cisplatin-induced kidney damage, and an application of eucommia extract in combination with cisplatin in the preparation of an anti-tumor drug; to overcome the cisplatin nephrotoxicity problem existing in the prior art and to enhance the anti-tumor efficacy of cisplatin.

[0005] In order to solve the above technical problems, the present invention specifically provides the following technical solutions: The invention discloses a pharmaceutical composition, including an active substance and an auxiliary liquid; the active substance includes at least one of eucommia extract and cisplatin; the eucommia extract is extracted from eucommia bark by ethanol reflux and purified; the eucommia extract contains 1.5-5.5% by mass of pinoresinol diglucoside. The prepared eucommia extract contains the effective ingredient pinoresinol diglucoside, and has a significant enhanced protective effect on the cell activity of human proximal tubule epithelial cell line and HK2 cells induced by cisplatin, and also has a mitigating effect on the shrinkage and damage of cells, and can also downregulate the relative expression level of KIM-1, a renal injury marker induced by cisplatin, and effectively alleviate renal injury. The eucommia extract is applied to a mouse model of acute renal injury induced by cisplatin, and the plasma creatinine, blood urea nitrogen and ALT levels of mice can also be significantly reduced, and the acute renal injury induced by cisplatin can be significantly improved. At the same time, the eucommia extract is used in combination with cisplatin to improve renal injury, and also has an inhibitory effect on endometrial cancer cell Ishikawa cells, and the two have a good synergistic promotion effect in anti-tumor.

[0006] Preferably, the auxiliary liquid is a cell culture medium.

[0007] Preferably, when the active substance is Eucommia ulmoides extract, the amount of the Eucommia ulmoides extract in the auxiliary liquid is 10-875 μg / ml.

[0008] Preferably, when the active ingredients are Eucommia ulmoides extract and cisplatin, the amount of Eucommia ulmoides extract in the auxiliary liquid is 10-875 μg / ml, and the mass ratio of cisplatin to Eucommia ulmoides extract is 1:2.08-182.

[0009] Preferably, the preparation of the secondary extract comprises: The bark of Eucommia ulmoides is crushed, an ethanol-water solution is added for reflux extraction, and then filtered to obtain an extract solution I and a filter residue, an ethanol-water solution is added to the filter residue for secondary extraction, and filtered to obtain an extract solution II, and the extract solution I and the extract solution II are combined and concentrated to obtain a concentrated solution, and then the concentrated solution is purified through an adsorption and elution process to obtain an Eucommia ulmoides extract; the adsorption and elution process uses an AB-8 macroporous adsorption resin column.

[0010] Preferably, the ethanol aqueous solution consists of ethanol and water, and the volume ratio of the amount of ethanol used to the amount of water used is 1:0.33-1.22.

[0011] Preferably, the dosage ratio of Eucommia bark to the ethanol aqueous solution added to the Eucommia bark is 1 g: 3-10 ml.

[0012] Preferably, the eluent used in the elution process is water and ethanol aqueous solution, the ethanol aqueous solution consists of ethanol and water, and the volume ratio of the amount of ethanol used to the amount of water used is 1:0.33-1.22.

[0013] Preferably, the volume ratio of the concentrated solution to the water used for elution is 1:1-2, and the volume ratio of the concentrated solution to the ethanol-water solution used for elution is 1:2-3.

[0014] Preferably, the reflux extraction time is 0.8-2.5h, and the secondary extraction time is 0.8-2.5h The invention discloses a method for preparing an eucommia extract, comprising: First, the bark of Eucommia ulmoides is crushed, and then an ethanol-water solution is added for reflux extraction for 0.8-2.5 hours, and then filtered to obtain an extract solution I and a filter residue, and an ethanol-water solution is added to the filter residue for secondary extraction for 0.8-2.5 hours, and filtered to obtain an extract solution II, and the extract solution I and the extract solution II are combined and concentrated to obtain a concentrated solution, and then the concentrated solution is adsorbed on an AB-8 macroporous adsorption resin column, and then purified through an elution process to obtain the Eucommia ulmoides extract.

[0015] Preferably, the ethanol aqueous solution consists of ethanol and water, and the volume ratio of the amount of ethanol used to the amount of water used is 1:0.33-1.22.

[0016] Preferably, the dosage ratio of Eucommia bark to the ethanol aqueous solution added to the Eucommia bark is 1 g: 3-10 ml.

[0017] Preferably, the eluent used in the elution process is water and ethanol aqueous solution, the ethanol aqueous solution consists of ethanol and water, and the volume ratio of the amount of ethanol used to the amount of water used is 1:0.33-1.22.

[0018] Preferably, the volume ratio of the concentrated solution to the water used for elution is 1:1-2, and the volume ratio of the concentrated solution to the ethanol-water solution used for elution is 1:2-3.

[0019] The present invention discloses a method for preparing a pharmaceutical composition, comprising: The active substance is added to the auxiliary solution to obtain a pharmaceutical composition.

[0020] Preferably, the active ingredient is at least one of Eucommia ulmoides extract and cisplatin.

[0021] Preferably, when the active substance is Eucommia ulmoides extract, the amount of Eucommia ulmoides extract added to the auxiliary liquid is 10-875 μg / ml.

[0022] Preferably, when the active ingredients are eucommia extract and cisplatin, the amount of eucommia extract added to the auxiliary liquid is 10-875 μg / ml, and the mass ratio of cisplatin to eucommia extract is 1:2.08-182.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention proposes the use of Eucommia ulmoides extract in the preparation of a drug for treating cisplatin-induced renal injury. The present invention first prepares Eucommia ulmoides extract, extracts Eucommia ulmoides peels through ethanol reflux extraction to obtain an extract, and the extract is adsorbed and purified using an AB-8 macroporous adsorption resin column to obtain Eucommia ulmoides extract. The prepared Eucommia ulmoides extract contains an effective ingredient, pinoresinol diglucoside; the Eucommia ulmoides extract is used in cisplatin-induced HK2 cells, which can significantly enhance the viability of HK2 cells and has a protective effect on HK2 cells; and can alleviate HK2 cell shrinkage injury; it has a down-regulating effect on the relative expression level of cisplatin-induced renal injury marker KIM-1, and can effectively alleviate renal injury. The Eucommia ulmoides extract is applied to a mouse model of acute renal injury induced by cisplatin, and can also significantly reduce the plasma creatinine, blood urea nitrogen and ALT levels of mice, and significantly improve cisplatin-induced acute renal injury. At the same time, the Eucommia ulmoides extract is used in combination with cisplatin to improve renal injury, and also has an inhibitory effect on endometrial cancer cell Ishikawa cells, and has a good synergistic anti-tumor cell effect. The eucommia extract of the present invention is a traditional Chinese medicine extract. Compared with artificially synthesized drugs, it has a comprehensive mechanism of action of multiple components, multiple pathways and multiple targets, has better therapeutic effects, lower side effects and better safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0025] Figure 1 This is a graph showing the effect of the Eucommia ulmoides extract prepared in Example 1 on the cell viability of HK2 cells; Figure 2 This is a graph showing the effect of the Eucommia ulmoides extract prepared in Example 2 on the cell viability of HK2 cells; Figure 3 This is a graph showing the effect of cisplatin on the cell viability of HK2 cells; Figure 4 This is a graph showing the effect of the Eucommia ulmoides extract prepared in Example 1 on the HK2 cell viability induced by cisplatin; Figure 5 This is a graph showing the effect of the Eucommia ulmoides extract prepared in Example 2 on the HK2 cell viability induced by cisplatin; Figure 6 This is the effect of Eucommia ulmoides extract on HK2 cell morphology induced by cisplatin; Figure 7 This is the scoring diagram of the effect of Eucommia ulmoides extract on cisplatin-induced HK2 cell damage; Figure 8This is the detection diagram of the effect of Eucommia ulmoides extract on the expression of KIM-1 in HK2 cells induced by cisplatin; Fig. 9 This is the effect of Eucommia ulmoides extract on the relative expression level of KIM-1 in HK2 cells induced by cisplatin; Fig.10 This is a test chart showing the effect of Eucommia ulmoides extract on biochemical indices of cisplatin-induced renal injury in mice; Fig.11 This is the H&E staining image of the effect of Eucommia ulmoides extract on cisplatin-induced renal injury in mice; Fig.12 This is a scoring diagram of the effect of Eucommia ulmoides extract on cisplatin-induced renal tubular injury in mice; Fig.13 This is a graph showing the effect of Eucommia ulmoides extract on the cell viability of human endometrial adenocarcinoma cell Ishikawa; Fig.14 This is a graph showing the effect of Eucommia ulmoides extract combined with cisplatin on the cell viability of human endometrial adenocarcinoma cell Ishikawa cells. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] The following first describes the concepts involved in the present application in conjunction with the accompanying drawings. It should be noted that the following description of each concept is only to make the content of the present application easier to understand, and does not limit the scope of protection of the present application; at the same time, the embodiments and features in the embodiments of the present application can be combined with each other in the absence of conflict. The present application will be described in detail with reference to the accompanying drawings and in conjunction with the embodiments.

[0028] Embodiment 1: Preparation of Eucommia ulmoides extract: First, pulverize the Eucommia ulmoides bark, then add ethanol aqueous solution to the Eucommia ulmoides bark and reflux for extraction for 1 hour, filter and separate to obtain extract I and filter residue, add ethanol aqueous solution to the filter residue and reflux for extraction for 1 hour, filter to obtain extract II, combine extract I and extract II, and finally concentrate and dry to obtain Eucommia ulmoides extract. The ethanol aqueous solution is composed of ethanol and water, the volume ratio of ethanol to water is 1:1, and the ratio of Eucommia ulmoides bark to the ethanol aqueous solution added to the Eucommia ulmoides bark is 1g:4.5ml.

[0029] Embodiment 2: Preparation of Eucommia ulmoides extract: First, crush the Eucommia ulmoides bark, then add ethanol aqueous solution to the Eucommia ulmoides bark and reflux extract for 1 hour, filter and separate to obtain extract I and filter residue, add ethanol aqueous solution to the filter residue and reflux extract for 1 hour, filter to obtain extract II, combine extract I and extract II, and concentrate to obtain a concentrated solution. The concentrated solution is passed through an AB-8 macroporous adsorption resin column, first eluted with water, then eluted with ethanol aqueous solution, and the eluate is collected. The eluate is then concentrated, washed with water, and dried to obtain the Eucommia ulmoides extract. The ethanol aqueous solution is composed of ethanol and water, the volume ratio of ethanol to water is 1:0.67, the ratio of Eucommia ulmoides bark to the ethanol aqueous solution added to the Eucommia ulmoides bark is 1g:4.5ml, the volume ratio of the concentrated solution to the water used for elution is 1:1.67, and the volume ratio of the concentrated solution to the ethanol aqueous solution used for elution is 1:2.33.

[0030] Embodiment 3: Preparation of the pharmaceutical composition: The pharmaceutical composition is obtained by adding the Eucommia ulmoides extract to a cell culture medium, wherein the amount of the Eucommia ulmoides extract added to the cell culture medium is 450 μg / ml, and the cell culture medium is a DMEM / F12 complete medium purchased from Thermo Fisher Scientific Inc.

[0031] Embodiment 4: Preparation of the pharmaceutical composition: The eucommia extract and cisplatin were added to a cell culture medium to obtain a pharmaceutical composition, wherein the amount of the eucommia extract added to the cell culture medium was 450 μg / ml, and the amount of cisplatin added to the cell culture medium was 16 μM, that is, the mass ratio of cisplatin to the eucommia extract was 1:93.59, and the cell culture medium was DMEM / F12 complete medium purchased from Thermo Fisher Scientific Inc.

[0032] Embodiment 5: Preparation of the pharmaceutical composition: The pharmaceutical composition was obtained by adding Eucommia ulmoides extract and cisplatin to a cell culture medium, wherein the amount of Eucommia ulmoides extract added to the cell culture medium was 112.5 μg / ml, and the amount of cisplatin added to the cell culture medium was 16 μM, that is, the mass ratio of cisplatin to Eucommia ulmoides extract was 1:23.4, and the cell culture medium was DMEM / F12 complete medium purchased from Thermo Fisher Scientific Inc.

[0033] Experimental Example 1: Determination of the content of pinoresinol diglucoside in the Eucommia ulmoides extract, with pinoresinol diglucoside as a control, the Eucommia ulmoides extract obtained in Examples 1 and 2 was subjected to HPLC detection to calculate the content of pinoresinol diglucoside. Content of pinoresinol diglucoside (%) = mass of pinoresinol diglucoside in the Eucommia ulmoides extract / mass of the Eucommia ulmoides extract × 100%.

[0034] Table 1 Determination results of pinoresinol diglucoside content

[0035] The results are shown in Table 1. The content of pinoresinol diglucoside in the Eucommia ulmoides extract prepared in Example 1 was 2%, and the content of pinoresinol diglucoside in the Eucommia ulmoides extract prepared in Example 2 was 5%.

[0036] Experimental Example 2: Renal proximal tubule epithelial cells are considered to be the target cells for cisplatin-induced nephrotoxicity, so immortalized renal proximal tubule epithelial cell line HK2 was used as the experimental cells. To determine the toxicity of Eucommia ulmoides extract on human proximal tubule epithelial cell line, HK2 cells, 5×10 cells in the logarithmic growth phase were first collected. 3 Cells were inoculated into the culture plate at a concentration of 100 cells / well, and cell culture medium was added for culture. Eucommia extract was then added to each experimental group, and a blank group (Con) without cells and Eucommia extract and a negative control group (CIS) without Eucommia extract were set up. After incubation for 48 hours, CCK-8 solution was added and incubated in a cell culture incubator for 1.5 hours. The OD value was measured at 490nm, and the cell viability was statistically calculated. The eucommia extract includes the eucommia extract prepared in Example 1 and Example 2, the addition amount of the eucommia extract prepared in Example 1 is 100, 200, 300, 400, 500, 600, 700 and 800 μg / ml, the addition amount of the eucommia extract prepared in Example 2 is 65.5, 131.1, 163.8, 204.8, 256, 320, 400 and 500 μg / ml, the volume ratio of the use amount of CCK-8 solution to the use amount of cell culture medium is 1:10, and the cell culture medium is DMEM / F12 complete culture medium purchased from Thermo Fisher Scientific Inc.

[0037] The results of the effects of different addition amounts of Eucommia ulmoides extract prepared in Example 1 on HK2 cell viability are as follows: Figure 1 As shown in the figure, when the amount of the Eucommia extract prepared in Example 1 was 100-700 μg / ml, the HK2 cell viability did not decrease, that is, there was no toxic side effect; when the amount of the Eucommia extract was 800 μg / ml, the cell viability decreased. The results of the effects of different amounts of the Eucommia extract prepared in Example 2 on the HK2 cell viability are shown in the figure. Figure 2 As shown, when the amount of the Eucommia extract prepared in Example 2 is 400 μg / ml, the effect on HK2 cell viability is still not obvious; when the amount of the Eucommia extract is increased to 500 μg / ml, HK2 cell viability will be inhibited. The maximum amount of the Eucommia extract with a pinoresinol diglucoside content of 2% and the Eucommia extract with a pinoresinol diglucoside content of 5% that has no inhibition on HK2 cell viability is about 2 times. The amount of Eucommia extract used needs to be within an appropriate range.

[0038] Experimental Example 3: Cisplatin-induced acute kidney injury model was established. Immortalized renal proximal tubule epithelial cell line HK2 was used as experimental cells. First, 5×10 cells in logarithmic growth phase were collected. 3 Cells were inoculated in a culture plate at a concentration of 100 μM / well, and cell culture medium was added for culture. Then, cisplatin was added to each experimental group, and a blank group (Con) without cells and cisplatin and a negative control group (CIS) without cisplatin were set up. After 48 hours of incubation, CCK-8 solution was added and incubated in a cell culture incubator for 1.5 hours. The OD value was measured at 490 nm, and the cell viability and IC50 value were statistically calculated. The amount of cisplatin added was 2, 4, 8, 10, 16, 24, 36 and 54 μM, the volume ratio of the amount of CCK-8 solution used to the amount of cell culture medium used was 1:10, and the cell culture medium was DMEM / F12 complete medium purchased from Thermo Fisher Scientific.

[0039] The results are as follows Figure 3 As shown, the IC50 value of cisplatin's inhibitory effect on HK2 cells was 33.36 μM; when the amount of cisplatin added was 16 μM, the cell viability was 61.74%, and 16 μM was used as the amount of cisplatin added to establish the cisplatin-induced acute kidney injury model.

[0040] Experimental Example 4: The protective effect of Eucommia ulmoides extract on cisplatin-induced acute renal injury and the effect on cisplatin-induced HK2 cell viability were determined. First, 5×10 cells in the logarithmic growth phase were collected. 3 The cells were inoculated into the culture plate at a concentration of 100 cells / well, and the cell culture medium was added for culture. Eucommia extract and cisplatin were then added to each experimental group, and a blank group (Con) without cells, Eucommia extract and cisplatin and a negative control group (CIS) without Eucommia extract were set up. After 48 hours of incubation, CCK-8 solution was added and incubated in a cell culture incubator for 1.5 hours. The OD value was measured at 490 nm, and the cell viability and protective effect were statistically calculated. Protective effect = cisplatin-induced HK2 cell viability after Eucommia extract addition − cisplatin-induced HK2 cell viability. The eucommia extract includes the eucommia extract prepared in Example 1 and Example 2, the added amount of the eucommia extract prepared in Example 1 is 300.7, 345.9, 397.7, 457.4, 526, 604.9, and 695.7 μg / ml, the added amount of the eucommia extract prepared in Example 2 is 91.9, 163.8, 204.8, 256, 320 and 400 μg / ml, the added amount of cisplatin is 16 μM, the volume ratio of the amount of CCK-8 solution used to the amount of cell culture medium used is 1:10, and the cell culture medium is DMEM / F12 complete culture medium purchased from Thermo Fisher Scientific Inc.

[0041] The results of the effects of different addition amounts of the Eucommia ulmoides extract prepared in Example 1 on the viability of HK2 cells induced by cisplatin are as follows: Figure 4 As shown in the figure, the addition of the Eucommia extract prepared in Example 1 has a significant protective effect on the viability of HK2 cells induced by cisplatin. When the amount of Eucommia extract added is 450 μg / ml, it is the optimal amount, and the protective effect is 13.16%. The results of the effects of different amounts of Eucommia extract prepared in Example 2 on the viability of HK2 cells induced by cisplatin are shown in the figure. Figure 5 As shown, when the amount of the Eucommia extract prepared in Example 2 is 200 μg / ml, it is the optimal amount, and the protective effect is 7.088%. When the amount of the Eucommia extract is increased to 400 μg / ml, the HK2 cell viability induced by cisplatin is inhibited, indicating that the amount of the Eucommia extract should be within a suitable range, and excessive use will inhibit the HK2 cell viability induced by cisplatin. Based on the size of the protective effect, the Eucommia extract prepared in Example 1 was selected for subsequent preparation of drugs for treating cisplatin renal injury and verification of the protective effect.

[0042] Experimental Example 5: To determine the effect of Eucommia ulmoides extract on the morphology of HK2 cells induced by cisplatin, 5×10 cells in the logarithmic growth phase were collected. 3 The concentration of 1000 cells / well was inoculated in a culture plate, and cell culture medium was added for culture. A total of six groups were divided into the first group, a control group (Con) without Eucommia extract and cisplatin, the second group added high-dose Eucommia extract (H_EUO), the third group added cisplatin (CIS), the fourth group added low-dose Eucommia extract and cisplatin (CIS+L_EUO), the fifth group added medium-dose Eucommia extract and cisplatin (CIS+M_EUO), and the sixth group added high-dose Eucommia extract and cisplatin (CIS+H_EUO). After incubation and culture for 48 hours, the morphology of HK2 cells was observed and the HK2 cell injury score was calculated. The Eucommia extract is the Eucommia extract prepared in Example 1, the addition amount of the low-dose Eucommia extract is 112.5 μg / ml, the addition amount of the medium-dose Eucommia extract is 225 μg / ml, the addition amount of the high-dose Eucommia extract is 450 μg / ml, the addition amount of cisplatin is 16 μM, and the cell culture medium is DMEM / F12 complete medium purchased from Thermo Fisher Scientific.

[0043] HK2 cell morphology results Figure 6As shown in the figure, compared with the control group without Eucommia extract and cisplatin, the HK2 cell morphology of the group with only high-dose Eucommia extract did not change significantly, and the HK2 cells of the group with only cisplatin showed shrinkage morphology changes; compared with the group with cisplatin, the HK2 cell morphology of the groups with low, medium and high doses of Eucommia extract and cisplatin showed dependent recovery according to the dose of Eucommia extract, indicating that Eucommia extract can effectively alleviate the shrinkage of HK2 cell morphology induced by cisplatin. The HK2 cell injury score results are shown in the figure. Figure 7 As shown, high-dose Eucommia ulmoides extract has no obvious toxic and inhibitory effect on HK2 cells, and can effectively alleviate cisplatin-induced HK2 cell damage.

[0044] Experimental Example 6: Effect of Eucommia ulmoides extract on the relative expression level of KIM-1, a marker of renal injury. The higher the relative expression level of KIM-1, the more severe the renal injury. The relative expression level of KIM-1 was detected by Western blot method using GAPDH as an internal reference. The group setting was the same as that of Experimental Example 5.

[0045] The relative expression level of KIM-1 was shown in Figure 8 and Fig. 9 As shown, compared with the control group without adding Eucommia extract and cisplatin, the group adding only high-dose Eucommia extract had no obvious effect on the relative expression level of KIM-1, and the group adding cisplatin significantly increased the relative expression level of KIM-1; compared with the group adding cisplatin, the groups adding low, medium and high doses of Eucommia extract and cisplatin can effectively reduce the relative expression level of KIM-1 according to the dosage of Eucommia extract.

[0046] Experimental Example 7: Biochemical indices related to mouse kidney and liver were detected, including plasma creatinine, blood urea nitrogen and ALT levels. C57BL / 6 mice were randomly divided into 6 groups (n=10 / group) according to body weight and administered by gavage. The first group was a control group (Con) without administration of Eucommia extract and cisplatin, the second group was administered with high-dose Eucommia extract (H_EUO), the third group was administered with cisplatin (CIS), the fourth group was administered with low-dose Eucommia extract and cisplatin (CIS+L_EUO), the fifth group was administered with medium-dose Eucommia extract and cisplatin (CIS+M_EUO), and the sixth group was administered with high-dose Eucommia extract and cisplatin (CIS+H_EUO). C57BL / 6 mice were purchased from Shanghai Slake Experimental Animal Co., Ltd., and were 6-8 weeks old.

[0047] First, the group given Eucommia extract was measured for body weight and administration once a day, and the administration was continued for 3 days. The first group and the third group were given an equal amount of normal saline. After 2 hours of administration on the third day, a single intraperitoneal injection of cisplatin was given to the group given cisplatin to induce the construction of an AKI mouse model, and the first group and the second group were intraperitoneally injected with an equal amount of normal saline. After 24 hours, the body weight was measured, the mice were anesthetized by isoflurane inhalation, the heart was blooded, and the mice were killed by cervical dislocation to take kidney tissue. Wherein C57BL / 6 mice were fasted for 12 hours before sampling, and 0.1% DMSO was contained in normal saline. The Eucommia extract was the Eucommia extract prepared by Example 1, and the dosage of low-dose Eucommia extract was 125mg / kg, the dosage of medium-dose Eucommia extract was 250mg / kg, and the dosage of high-dose Eucommia extract was 500mg / kg. The results of acute toxicity test showed that Eucommia extract had no significant toxic effects on mice at a dosage of 500mg / kg, and the dosage of cisplatin was 20mg / Kg.

[0048] The results of plasma creatinine, blood urea nitrogen and ALT levels are as follows Fig.10 As shown, compared with the control group not administered with Eucommia extract and cisplatin, the plasma creatinine, blood urea nitrogen and ALT levels of the group only administered with high dose of Eucommia extract did not change significantly, while the plasma creatinine, blood urea nitrogen and ALT levels of the group administered with cisplatin were significantly increased; compared with the group administered with cisplatin, the plasma creatinine, blood urea nitrogen and ALT levels of the groups administered with low, medium and high doses of Eucommia extract and cisplatin were significantly decreased, indicating that Eucommia extract can effectively alleviate cisplatin-induced kidney and liver damage.

[0049] Experimental Example 8: The degree of renal injury in different groups of mice in Experimental Example 7 was detected by H&E staining. Fig.11 As shown in the figure, compared with the control group without eucommia extract and cisplatin, the mice in the group treated with cisplatin showed more severe renal damage, and the brushing edge of the renal tubule disappeared; compared with the group treated with cisplatin, the degree of renal damage in the groups treated with low, medium and high doses of eucommia extract and cisplatin was significantly reduced in a dose-dependent manner. Fig.12 As shown, it was shown that Eucommia ulmoides extract can effectively alleviate cisplatin-induced renal injury.

[0050] Embodiment 9: The inhibitory effect of Eucommia ulmoides extract on human endometrial adenocarcinoma Ishikawa cells and its anti-tumor effect. First, 5×10 cells in the logarithmic growth phase were collected. 3The concentration of cells / well was inoculated in a culture plate, and cell culture medium was added for culture. Then, Eucommia extract was added to each experimental group, and a blank group (Con) without cells and Eucommia extract and a negative control group (CIS) without Eucommia extract were set. After incubation for 48 hours, CCK-8 solution was added and incubated in a cell culture incubator for 1.5 hours, and the OD value was measured at 490nm, and the cell viability and IC50 value were statistically calculated. The Eucommia extract was the Eucommia extract prepared in Example 1, and the amount of Eucommia extract added was 12.5, 25, 50, 100, 200, 400, 800 and 1600 μg / ml, the volume ratio of the amount of CCK-8 solution used to the amount of cell culture medium used was 1:10, and the cell culture medium was DMEM culture medium purchased from Thermo Fisher Scientific.

[0051] The results are as follows Fig.13 As shown, the Eucommia extract showed an inhibitory effect on the human endometrial adenocarcinoma Ishikawa cells used in the experiment, and the effect was dose-dependent. The IC50 value of the Eucommia extract for the human endometrial adenocarcinoma Ishikawa cells was 565.2 μg / ml, and it had no toxic effect on normal human renal tubular epithelial cells HK2.

[0052] Embodiment 10: The inhibitory effect of Eucommia ulmoides extract combined with cisplatin on human endometrial adenocarcinoma Ishikawa cells and their anti-tumor effects. First, 5×10 cells in the logarithmic growth phase were collected. 3 The cells were inoculated in a culture plate at a concentration of 100 cells / well, and cultured in cell culture medium. The cells were divided into six groups: the first group was a control group (Con) without Eucommia extract and cisplatin, the second group was added with high-dose Eucommia extract (H_EUO), the third group was added with cisplatin (CIS), the fourth group was added with low-dose Eucommia extract and cisplatin (CIS+L_EUO), the fifth group was added with medium-dose Eucommia extract and cisplatin (CIS+M_EUO), and the sixth group was added with high-dose Eucommia extract and cisplatin (CIS+H_EUO). After incubation for 48 hours, CCK-8 solution was added and incubated in a cell culture incubator for 1.5 hours. The OD value was measured at 490 nm, and the cell viability was statistically calculated. The eucommia extract is the eucommia extract prepared in Example 1, the added amount of the low-dose eucommia extract is 112.5 μg / ml, the added amount of the medium-dose eucommia extract is 225 μg / ml, the added amount of the high-dose eucommia extract is 450 μg / ml, the added amount of cisplatin is 16 μM, the volume ratio of the used amount of CCK-8 solution to the used amount of cell culture medium is 1:10, and the cell culture medium is DMEM culture medium purchased from Thermo Fisher Scientific Inc.

[0053] The results are as follows Fig.14As shown, Eucommia extract can promote the inhibitory effect of cisplatin on endometrial cancer cell Ishikawa cells in a dose-dependent manner, and the two have a synergistic effect. Eucommia extract and first-line chemotherapy drug cisplatin form a combined prescription drug, which can effectively inhibit tumors.

[0054] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0055] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. A pharmaceutical composition comprising an active substance and an auxiliary liquid; the active substance comprises at least one of an eucommia extract and cisplatin; the eucommia extract is obtained by refluxing extraction of eucommia bark with an ethanol aqueous solution and purifying the eucommia extract; the eucommia extract contains 1.5-5.5% by mass of pinoresinol diglucoside.

2. A pharmaceutical composition according to claim 1, characterized in that: The auxiliary liquid is a cell culture medium.

3. A pharmaceutical composition according to claim 1, characterized in that: When the active substance is eucommia extract, the amount of eucommia extract in the auxiliary liquid is 10-875 μg / ml.

4. A pharmaceutical composition according to claim 1, characterized in that: When the active substances are eucommia extract and cisplatin, the amount of eucommia extract in the auxiliary liquid is 10-875 μg / ml, and the mass ratio of cisplatin to eucommia extract is 1:2.08-182.

5. A pharmaceutical composition according to claim 1, characterized in that: The preparation of the Eucommia ulmoides extract comprises: The bark of Eucommia ulmoides is crushed, an ethanol-water solution is added for reflux extraction, and then filtered to obtain an extract solution I and a filter residue, an ethanol-water solution is added to the filter residue for secondary extraction, and filtered to obtain an extract solution II, and the extract solution I and the extract solution II are combined and concentrated to obtain a concentrated solution, and then the concentrated solution is purified through an adsorption and elution process to obtain an Eucommia ulmoides extract; the adsorption and elution process uses an AB-8 macroporous adsorption resin column.

6. A pharmaceutical composition according to claim 5, characterized in that: The ethanol aqueous solution consists of ethanol and water, and the volume ratio of the amount of ethanol used to the amount of water used is 1:0.33-1.

22.

7. A pharmaceutical composition according to claim 5, characterized in that: The dosage ratio of the eucommia bark to the ethanol aqueous solution added to the eucommia bark is 1g:3-10ml.

8. A pharmaceutical composition according to claim 5, characterized in that: The eluent used in the elution process is water and ethanol aqueous solution, the ethanol aqueous solution is composed of ethanol and water, and the volume ratio of the used amount of ethanol to the used amount of water is 1:0.33-1.

22.

9. A pharmaceutical composition according to claim 5, characterized in that: The volume ratio of the concentrated solution to the water used for elution is 1:1-2, and the volume ratio of the concentrated solution to the ethanol-water solution used for elution is 1:2-3.

10. A pharmaceutical composition according to claim 5, characterized in that: The reflux extraction time is 0.8-2.5h, and the secondary extraction time is 0.8-2.5h.

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